Comparative Proteomic Analysis of Supportive and Unsupportive Extracellular Matrix Substrates for Human Embryonic Stem Cell Maintenance

Human embryonic stem cells (hESCs) are pluripotent cells that have indefinite replicative potential and the ability to differentiate into derivatives of all three germ layers. hESCs are conventionally grown on mitotically inactivated mouse embryonic fibroblasts (MEFs) or feeder cells of human origin. In addition, feeder-free culture systems can be used to support hESCs, in which the adhesive substrate plays a key role in the regulation of stem cell self-renewal or differentiation. Extracellular matrix (ECM) components define the microenvironment of the niche for many types of stem cells, but their role in the maintenance of hESCs remains poorly understood. We used a proteomic approach to characterize in detail the composition and interaction networks of ECMs that support the growth of self-renewing hESCs. Whereas many ECM components were produced by supportive and unsupportive MEF and human placental stromal fibroblast feeder cells, some proteins were only expressed in supportive ECM, suggestive of a role in the maintenance of pluripotency. We show that identified candidate molecules can support attachment and self-renewal of hESCs alone (fibrillin-1) or in combination with fibronectin (perlecan, fibulin-2), in the absence of feeder cells. Together, these data highlight the importance of specific ECM interactions in the regulation of hESC phenotype and provide a resource for future studies of hESC self-renewal.     

FGF-23 Is a Negative Regulator of Prenatal and Postnatal Erythropoiesis

Abnormal blood cell production is associated with chronic kidney disease (CKD) and cardiovascular disease (CVD). Bone-derived FGF-23 (fibroblast growth factor-23) regulates phosphate homeostasis and bone mineralization. Genetic deletion of Fgf-23 in mice (Fgf-23^−/−) results in hypervitaminosis D, abnormal mineral metabolism, and reduced lymphatic organ size. Elevated FGF-23 levels are linked to CKD and greater risk of CVD, left ventricular hypertrophy, and mortality in dialysis patients. However, whether FGF-23 is involved in the regulation of erythropoiesis is unknown. Here we report that loss of FGF-23 results in increased hematopoietic stem cell frequency associated with increased erythropoiesis in peripheral blood and bone marrow in young adult mice. In particular, these hematopoietic changes are also detected in fetal livers, suggesting that they are not the result of altered bone marrow niche alone. Most importantly, administration of FGF-23 in wild-type mice results in a rapid decrease in erythropoiesis. Finally, we show that the effect of FGF-23 on erythropoiesis is independent of the high vitamin D levels in these mice. Our studies suggest a novel role for FGF-23 in erythrocyte production and differentiation and suggest that elevated FGF-23 levels contribute to the pathogenesis of anemia in patients with CKD and CVD.     

CV and NMR study on the reaction of Mo(VI) with 3,4-dihydroxybenzoic acid and ascorbic acid in aqueous solution

The gradual release of the ligand 3,4-dihydroxybenzoic acid (3,4-DHBA) from its molybdenum complex in the presence of ascorbic acid (AscA) in a weakly acidic aqueous solution (pH ∼ 3.5) is described. We observed that the formation of the 3,4-DHBA-semiquinone oxidation state and the semidehydroascorbate is a pre-requisite for the release of the 3,4-DHBA ligand. The interaction of these radicals leads at the same time to the further degradation of AscA resulting in, among other compounds, threonic acid which participates in the reaction with molybdenum. The comparison of the complexing ability indicated that threonic acid competes with protocatechuate, while ascorbic acid is a less good ligand for the Mo(VI). Solution studies on the reaction mechanism were performed by cyclic voltammetry, NMR spectroscopy and UV-Vis spectroscopy. Isolated precipitates were investigated by NMR spectroscopy. The antioxidant properties of 3,4-DHBA and AscA were also compared using the stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH).     

Stimulation of endocytosis and actin dynamics by Oskar polarizes the Drosophila oocyte

In Drosophila, localized activity of oskar at the posterior pole of the oocyte induces germline and abdomen formation in the embryo. Oskar has two isoforms, a short isoform encoding the patterning determinant and a long isoform of unknown function. Here, we show by immuno-electron microscopy that the two Oskar isoforms have different subcellular localizations in the oocyte: Short Oskar mainly localizes to polar granules, and Long Oskar is specifically associated with endocytic membranes along the posterior cortex. Our cell biological and genetic analyses reveal that Oskar stimulates endocytosis, and that its two isoforms are required to regulate this process. Furthermore, we describe long F-actin projections at the oocyte posterior pole that are induced by and intermingled with Oskar protein. We propose that Oskar maintains its localization at the posterior pole through dual functions in regulating endocytosis and F-actin dynamics.     

Dipstick-type biosensor for visual detection of DNA with oligonucleotide-decorated colored polystyrene microspheres as reporters

In recent years, there is a continuously growing interest in the development of biosensors for rapid, simple and inexpensive DNA tests suitable for the small laboratory or for on-site testing. Detection is accomplished through electrochemical, optical or gravimetric transduction. We report on the development of disposable dipstick-type DNA biosensors that employ oligonucleotide-decorated colored polystyrene microspheres as reporters and enable visual detection of DNA sequences without the use of instrumentation. The biosensors have been designed to detect DNA molecules that contain both, a biotin moiety and a segment that is complementary to the oligonucleotide attached on the surface of blue or red microspheres. Capture of the hybrids by immobilized streptavidin at the test zone results in the formation of a colored line. The biosensors were applied to: (a) detection of single-stranded DNA, (b) detection of PCR-amplified double-stranded DNA and (c) genotyping of single nucleotide polymorphisms (SNP). The results were compared with sensors based on gold nanoparticle reporters. It is also demonstrated that the microspheres offer the potential for multicolor detection of specific DNA sequences.     

The anti-apoptotic protein HAX-1 interacts with SERCA2 and regulates its protein levels to promote cell survival

Cardiac contractility is regulated through the activity of various key Ca²⁺-handling proteins. The sarco(endo)plasmic reticulum (SR) Ca²⁺ transport ATPase (SERCA2a) and its inhibitor phospholamban (PLN) control the uptake of Ca²⁺ by SR membranes during relaxation. Recently, the antiapoptotic HS-1–associated protein X-1 (HAX-1) was identified as a binding partner of PLN, and this interaction was postulated to regulate cell apoptosis. In the current study, we determined that HAX-1 can also bind to SERCA2. Deletion mapping analysis demonstrated that amino acid residues 575–594 of SERCA2's nucleotide binding domain are required for its interaction with the C-terminal domain of HAX-1, containing amino acids 203-245. In transiently cotransfected human embryonic kidney 293 cells, recombinant SERCA2 was specifically targeted to the ER, whereas HAX-1 selectively concentrated at mitochondria. On triple transfections with PLN, however, HAX-1 massively translocated to the ER membranes, where it codistributed with PLN and SERCA2. Overexpression of SERCA2 abrogated the protective effects of HAX-1 on cell survival, after hypoxia/reoxygenation or thapsigargin treatment. Importantly, HAX-1 overexpression was associated with down-regulation of SERCA2 expression levels, resulting in significant reduction of apparent ER Ca²⁺ levels. These findings suggest that HAX-1 may promote cell survival through modulation of SERCA2 protein levels and thus ER Ca²⁺ stores.     

STAT-Related Profiles Are Associated with Patient Response to Targeted Treatments in Locally Advanced SCCHN

The anti-epidermal growth factor receptor antibody cetuximab (Erbitux, CTX) is currently used for the treatment of locally advanced squamous cell carcinoma of the head and neck (LA-SCCHN), as yet with modest effectiveness, prompting for the identification of response predictors to this treatment and for the targeting of additional pathways implicated in this disease. Within this scope, we investigated the effect of SRC/STAT pathway components on LA-SCCHN patient outcome. SRC, STAT1, STAT3, STAT5A, STAT5B, ANXA1, CAV1, IGFBP2, EPHA2, EPHB2, and MSN relative gene expression, as well as Stat protein activation, were assessed on LA-SCCHN tumor tissues from 35 patients treated with combined radiotherapy (RT) and CTX-based regimens. Stat1, Stat3, and Stat5 proteins were usually found activated in neoplastic nuclei (70.4%, 85.7%, and 70.8%, respectively). Activated Stat3 and Stat5 were associated with each other (P = .017) and with a CAV1(high)/MSN(high)/IGFBP2(low) profile. All patients with tumors expressing high STAT5A/EPHA2 experienced a complete response on RT-CTX-based treatments (12/15 complete responders, P < .0001) and a longer progression-free survival (P = .024). Few tumors expressed high ANXA1/CAV1/EPHA2 and low IGFBP2, a putative dasatinib response-related profile, whereas high ANXA1 was associated with shorter overall survival (P = .003). In conclusion, Stat activation is common in LA-SCCHN, where overexpression of STAT5A and EPHA2 may predict for response to RT-CTX treatments. The STAT5A/EPHA2 profile seems of particular interest for validation in larger cohorts and in multiple tumor types because markers for the positive selection of patients to benefit from CTX-containing treatments are currently lacking.